Summary: Potency of VIG Products: Development of in vivo models to assess VIG potency We have recently observed that VIG preparations which appear to have equivalent PRN titers, have different ability to neutralize vaccinia in vivo in SCID mice, suggesting that PRN assays as currently performed are unable to measure an important marker(s) of efficacy. Vaccinia viruses occur in two infectious forms. The intracellular mature form (IMV) remains inside cells until lysis, and accounts for the majority of virus. The extracellular enveloped form buds from live cells, is surrounded by an extra host-derived lipid envelope, and contains host and virus proteins that are absent from IMV. Since the 1970's, it has been recognized that distinct subsets of antibodies neutralize each form; furthermore that antibodies to EEV as well as IMV are required for active and passive immune protection. The PRN, by nature of the viral preparation used, measures antibodies against IMV, but not EEV, and this could be one possible cause of the discrepancy between the in vivo and in vitro neutralization data. Other possibilities include effects of complement fixation, and antibody-dependent cellular cytotoxicity induction by VIG preparations, which are also not measured in vitro, but may differ among immune globulin preparations. The SCID mouse model, while useful in preliminary experiments, has several drawbacks, both practical and theoretical. All experiments to date have been performed by mixing antibodies and virus in vitro, followed by i.p. injection of the combined preparations. While this demonstrates differences among products (which are not detected by PRN or HPLC), the model has been criticized because it does not simulate the likely human clinical situation. Indications for VIG in humans have been prophylaxis against complications (in eczema, immunodeficiency, pregnancy), and treatment of disseminated infection after vaccination (progressive vaccinia and eczema vaccinatum). It has also been suggested that smallpox attack rates among primary contacts can be diminished by VIG. Thus more relevant models would use different routes of exposure (e.g. intradermal, mucosal), would measure in vivo neutralization of virus by VIG, and would compare the ability of products to provide prophylaxis and treat extensive vaccinia infection. Furthermore the lethality assay in SCID mice is prolonged (28 days minimum), and requires extensive monitoring twice daily, 7 days/week. A new, rapid, relevant animal model could be used to develop optimal in vitro assays, or combinations of assays; to compare products; and to determine product characteristics that are required for efficacy. We plan to establish in vivo models of widespread vaccinia infection, using relevant exposure methods, in mice. First, a new mouse model will be developed to simulate human cutaneously acquired infection that has disseminated in immunodeficient patients. Immunodeficient mice (SCID, and IL-15 KO) will be injected with doses of vaccinia (Wyeth strain, and Dryvax, 103 to 106 PFU) i.d. (flank), and by scarification, to determine dose-response to virus, and time to lethality. IL-15 KO mice are susceptible to severe vaccinia infection, due to lack of NK cells and inability to generate CTL. SCID mice lack B and T cells, and are thus unable to generate either cellular or humoral immunity, although they retain NK function. A dose of virus and optimal mouse model will be selected using results of preliminary experiments, and mice will receive prophylaxis or treatment (in separate experiments) with VIG preparations (5- 50 mg/mouse) i.v. and/or i.p. The outcome for these experiments is lethality, however surrogate markers (e.g. weight loss and onset of other clinical symptoms; lesion size) will be explored in the course of study, in hopes of further curtailing the amount of time needed per experiment, and to diminish discomfort to mice. Pilot vaccinia infection studies will be performed in mice that are susceptible to atopic dermatitis. If these mice develop a syndrome analagous to eczema vaccinatum, VIG prophylaxis and treatment will also be tested in these strains. VIG products may be required for emergency use in case of mass vaccination or a smallpox attack. It is important to maintain current data on stability and potency of these products. VIG (Baxter 1994, the current IND product available for use), and VIGIV products (all lots) will be aliquotted and stored at the recommended storage temperature. We plan to assess each preparation by HPLC (which indicates fragmentation, and aggregation), by PRN assay, and B-galactosidase assay, using our new frozen CBER working standard as a control preparation. The PRN assay will be performed every 3 months for VIG, which is the current first-line preparation. VIG is eight years old, has been stored at 4o C, and contains fragments and aggregates. As the first-line preparation, VIG should be watched closely for signs of decline. We will test VIGIV's every 6 months for stability and potency. As assays evolve and become more specifically correlated with protection, we will perform additional in vivo and in vitro evaluations as they are deemed relevant.